Two species of alpha-lactalbumin, alpha-lactalbumin1 and alpha-lactalbumin2, were separated from rat milk and purified to homogeneity by gel filtration, followed by DEAE-cellulose chromatography, alpha-lactalbumin, shows a molecular weight of 21,500 as determined by sedimentaiion equilibrium analysis and sodium dodecyl sulfate-polyacrylamide gel analysis. Alpha-lactalbumin2 has a molecular weight of 16,000 measured by sedimentation analysis. Alpha-lactallumin2, however, exhibits abnormally high molecular weight of 22,500 on sodium dodecyl sulfate polyacrylamide gels. Both alpha-lactalbumins are active in lactose synthase assay and are glycoproteins containing 7 to 9% carbohydrate. Antiserum raised against alpha-lactalbumin1 cannot discriminate between the two species in a radioimmunoassay.